Treatment and prevention of hyperproliferative conditions in humans and antisense oligonucleotides inhibition of human replication-initiation proteins

ABSTRACT

Antisense oligonucleotides that inhibit expression of human replication-initiation protein as well as methods of preventing or treating hyperproliferative conditions using said oligonucleotides are disclosed. One aspect provides an antisense oligonucleotides that inhibits the expression of human replication-initiation protein and has a sequence complementary to at least a portion of a target sequence encoding a human replication-initiation gene. By administering a therapeutically effective amount of antisense oligonucleotides or by contacting the hyperproliferating cells with an effective amount of antisense oligonucleotides, expression of replication-initiation protein is inhibited. Methods of screening and testing active antisense oligonucleotides for their ability to inhibit gene expression are also disclosed.

REFERENCE TO SEQUENCE LISTING

[0001] Two copies of the “Sequence Listing” in computer readable form incompliance with 37 C.F.R. §1.821 to 1.825 are enclosed. The sequencelisting information recorded in computer readable form is identical tothe written on paper sequence listing, and incorporated herein byreference.

BACKGROUND

[0002] The present invention relates to treatment and prevention ofhyperproliferative conditions in humans and more particularly toantisense oligonucleotides complementary to human replication-initiationgenes which modulate DNA replication and cell proliferation in humans.This invention also relates to methods of using such oligonucleotides toinhibit the growth of tumor cells in mammals.

[0003] Cancer presents one of the most serious threats to human healthand life. There are approximately ten million new cancer cases and sevenmillion cancer-related deaths every year in the world. In fact, one inabout every four people has the probability of developing cancer in alifetime.

[0004] Some examples of anticancer drugs currently available arecytotoxins, DNA damaging agents, and inhibitors of oncogenic proteinsinvolved in signal transduction pathways for cell proliferation.However, few of the current anticancer drugs are effective or withoutside effects. Many of these drugs are not highly selective towardscancer cells and these drugs also damage normal cells or inhibit themetabolism and cellular functions of normal cells. Moreover, mostoncogenic signal transduction pathways are redundant in the cells, andtherefore, blocks to individual pathways can be bypassed by cancer cellsand inhibition of one or some of these pathways may not stop cancergrowth.

[0005] Antisense oligonucleotides are known to be able to inhibit geneexpression, and have been used in combination with chemotherapeuticagents in developing anticancer strategies in mouse xenographs (Geiger,T. et al., Anti-Cancer Drug Design, 13, 35-45 (1998), Del Bufalo, D. etal, British J. of Cancer, 74, 387-393 (1996)). Current theories suggestthat the activity of antisense oligonucleotides depends on the bindingof the oligonucleotides to the target nucleic acid (e.g. to at least aportion of a genomic region, gene or MRNA transcript thereof), thusdisrupting the function of the target, either by hybridization arrest orby destruction of target RNA by RNase H (the ability to activate RNase Hwhen hybridized to RNA). For example, antisense oligonucleotides bind tothe complementary sequence on a target MRNA nucleic acid sequence, thusactivating endogenous RNase H to cleave mRNA. Binding of antisenseoligonucleotides to MRNA may also interfere with translation of MRNAthereby reducing or eliminating production of a gene even if the mRNA isnot degraded (Milligan, J. F., et al, J. Med. Chem., 36, 1923-1927(1993)). However, known antisense oligonucleotides have not targetedhuman replication-initiation genes, nor demonstrated any efficacy ininhibiting expression of replication-initiation proteins in human cells.

[0006] Proteins involved in the initiation of DNA replication (i.e.,genome duplication) present excellent targets for cancer therapy.Initiation of DNA replication is controlled by the cis-acting DNAelements called replicators and the trans-acting initiation proteinsthat interact with the replicators. To date, several groups ofinitiation proteins required for eukaryotic DNA replication have beenidentified. These include ORC (origin recognition complex), Cdc6 (celldivision cycle), MCM (minichromosome maintenance), Cdc45 and Cdt1proteins (Takisawa, H., et al., Curr. Opin. Cell. Biol. 12, 690-696(2000)). ORC binds chromatin throughout the cell cycle, whereas thechromatin association of other groups is cell cycle-regulated(Leatherwood, J., Curr. Opin. Cell. Biol., 10, 742-748 (1998)). Some ofthe initiation proteins in humans, such as Cdc6 and MCM proteins, areexpressed in cancerous, but not in normal, non-dividing cells (Williams,G. H. et al., Proc. Natl. Acad. Sci. USA, 95, 14932-14937 (1998)). Genesequences encoding some of these proteins have been isolated (Williams,U.S. Pat. No. 5,851,821; Saha, P. et al., J. Biol. Chem., 50, 6075-6086(1990); Todorov, LT., et al., J. Cell Sci., 107, 253-265 (1994)).

SUMMARY

[0007] The present invention relates to treatment and prevention ofhyperproliferative conditions in humans and to antisenseoligonucleotides that inhibit expression of human replication-initiationprotein as well as methods of preventing or treating hyperproliferativeconditions using these oligonucleotides. Methods of screening andtesting active antisense oligonucleotides for their ability to inhibitgene expression are also disclosed.

[0008] One aspect provides an antisense oligonucleotides that inhibitsthe expression of human replication-initiation protein and has asequence complementary to at least a portion of a target sequence of ahuman replication-initiation gene.

[0009] Another aspect is directed toward a method of preventing ortreating hyperproliferative conditions by way of example but not limitedto cancer, angiogenesis or neovascularization. By administering atherapeutically effective amount of antisense oligonucleotides or bycontacting the hyperproliferating cells with a effective amount ofantisense oligonucleotides, expression of replication-initiation proteinis inhibited.

[0010] Yet another aspect is directed toward screening and testingantisense oligonucleotides that inhibit gene expression. One embodimentinvolves screening an antisense oligonucleotides or inhibition of geneexpression. After selecting one or more antisense oligonucleotides thatinhibited gene expression, modifying those selected antisenseoligonucleotides such that the oligonucleotides contains aphosphorothioate linkage between the first two nucleotides and aphosphorothioate linkage between the last two nucleotides of thesequence. The modified oligonucleotides are screened once again forinhibition of gene expression and the modified oligonucleotides theinhibited gene expression are further modified by replacing one or moreinternucleosidic linkages with phosphorothioate linkages.

[0011] These and other features of the claims will be appreciated fromreview of the following detailed description of the invention along withthe accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 illustrates the results from RT-PCR analysis of hCdc45,hCdc6, hMcm2 and the internal control β-actin gene for the cells treatedwith SEQ. ID. No. 16-18;

[0013]FIG. 2 illustrates the results from RT-PCR analysis of hCdc45,hCdc6, hMcm2 and the internal control β-actin gene for the cells treatedwith the SEQ. ID. Nos. 25-27;

[0014]FIG. 3 illustrates the results from RT-PCR analysis of hCdc45,hCdc6, hMcm2 and the internal control β-actin gene for the cells treatedwith the antisense oligonucleotides SEQ. ID. No. 1 or the controloligonucleotides;

[0015]FIG. 4 illustrates the results from Western blotting for hCdc6 inthe protein extracts from cells treated with SEQ. ID. Nos. 1, 4, and 6targeted to hCdc6;

[0016]FIG. 5 illustrates the results from Western blotting for Caspase-3(A) and PARP (B) in the protein extracts from cells treated with SEQ.ID. No. 25 targeted to hCdc45;

[0017]FIG. 6 illustrates the micrographs of the untreated cells andthose treated with liposome without oligonucleotides, the antisenseoligonucleotides SEQ. ID. No. 16 or the control oligonucleotides SEQ.ID. Nos. 17 and 18;

[0018]FIG. 7 illustrates the results of DNA fragmentation, which is anindication of apoptotic cell death induced by SEQ. ID. No. 25;

[0019]FIG. 8 illustrates the results of DNA fragmentation, as measuredby the TUNEL assay, in liver cancer cells, but not in normal liver cellsafter treatment with SEQ. ID. No. 16; and

[0020]FIG. 9 illustrates the results showing that antisenseoligonucleotides Seq. ID. Nos. 1, 16, and 20 reduced human cancer growthin nude mice xenographs.

DETAILED DESCRIPTION

[0021] Preferred embodiments of antisense oligonucleotides that inhibithuman replication-initiation protein expression as well as methods ofusing these antisense oligonucleotides to prevent or treathyperproliferation conditions are described in non-limiting detailbelow. Methods of screening and testing active antisenseoligonucleotides that inhibit gene expression are also described.

[0022] The claimed antisense oligonucleotides described by the presentclaims have sequences complementary to target nucleic acid sequencesencoding any portion of a human replication-initiation gene. The term“antisense” refers to the complementary relationship between anantisense oligonucleotides and its complementary nucleic acid target (towhich it hybridizes). An antisense oligonucleotides is formed by“targeting” an oligonucleotides to a chosen nucleic acid sequence. Inthis invention, the targeted sequence is any portion of an encodingsequence for a human replication-initiation protein. For example, thetargeted sequence may be a sequence encoding any portion of human hCdc6,hCdc45, hMcm2, hMcm3, hMcm4, hMcm5, hMcm6, hMcm7, hOrc1, hOrc2, hOrc3,hOrc4, hOrc5, hOrc6 and hCdt1 proteins. However, other humanreplication-initiation proteins may be used. In one embodiment, thetargeted sequence is SEQ ID Nos. 3, 6, 9, 12, 15, 18, 21, 24, or 27

[0023] The targeting process also includes determination of a site orsites within the nucleic acid sequence for the oligonucleotidesinteraction to occur such that inhibition of gene expression results.Once the target site or sites on the nucleic acid sequence have beenidentified, oligonucleotides are chosen that are sufficientlycomplementary to the target, i.e., hybridize sufficiently well and withsufficient specificity, which can be measured in ways which are routinein the art, for example, by Northern blot assay of mRNA expression, orreverse transcriptase PCR, Western blot, ELIS A assay of proteinexpression, or immunoprecipitation assay of protein expression. However,other methods are known in the art and may be used. Effects on cellproliferation or tumor cell growth can also be measured, as taught inthe examples below.

[0024] “Hybridization” refers to hydrogen bonding, also known asWatson-Crick base pairing, between complementary bases, usually onopposite nucleic acid strands or two regions of a nucleic acid strand.Guanine and cytosine are examples of complementary bases which are knownto form three hydrogen bonds between them. Adenine and thymine areexamples of complementary bases that form two hydrogen bonds betweenthem. The oligonucleotides is hybridized with sufficient specificitywhen a sufficient degree of complementarity effects stable and specificbinding between the DNA or RNA target and the oligonucleotides. Anoligonucleotides is specifically hybridizable when binding of theoligonucleotides to the target interferes with the normal function ofthe target molecule to cause a loss of utility, and there is asufficient degree of complementarity to avoid non-specific binding ofthe oligonucleotides to non-target sequences under conditions in whichspecific binding is desired, i.e., under physiological conditions in thecase of in vivo assays or therapeutic treatment or, in the case of invitro assays, under conditions in which the assays are conducted. Thefunctions of mRNA to be interfered with include all vital functions suchas, for example, translocation of the RNA to the site of proteintranslation, translation of protein from the RNA, splicing of the RNA toyield one or more mRNA species, and catalytic activity which may beengaged in by the RNA. Binding of specific protein(s) to the RNA mayalso be interfered with by antisense oligonucleotides hybridization tothe RNA. The overall effect of interference with mRNA function isinhibition of human replication-initiation proteins. Practitioners inthe art will appreciate that an oligonucleotides need not be 100%complementary to its target nucleic acid sequence to be specificallyhybridizable. For example, in one embodiment, the claimed antisenseoligonucleotides has a sequence that is 90% complementary to the targetsequence.

[0025] The term “antisense oligonucleotides” as used herein means anucleotide sequence that is complementary to the desired MRNA encodingany portion of human replication-initiation proteins. The antisenseoligonucleotides is complementary to any portion of humanreplication-initiation protein mRNA that effectively acts as a targetfor inhibiting human replication-initiation protein expression.

[0026] Practitioners in the art understand that mRNA includes not onlythe information to encode a protein using the three letter genetic code,but also associated ribonucleotides which form a region known as the5′-untranslated region, the 3′-untranslated region, the 5′ cap regionand intron/exon junction ribonucleotides. Thus, oligonucleotides may beformulated in accordance with this invention, which are targeted whollyor in part to these associated ribonucleotides as well as to theinformational ribonucleotides. The oligonucleotides may therefore bespecifically hybridizable with a transcription initiation site region, atranslation initiation codon region, a 5′ cap region, an intron/exonjunction, coding sequences, a translation termination codon region orsequences in the 5′- or 3′-untranslated region. Since, as is known inthe art, the translation initiation codon is typically 5′-AUG (intranscribed mRNA molecules; 5′-ATG in the corresponding DNA molecule),the translation initiation codon is also referred to as the “AUG codon,”the “start codon” or the “AUG start codon.” A minority of genes have atranslation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function invivo. Thus, the terms “translation initiation codon” and “start codon”can encompass many codon sequences, even though the initiator amino acidin each instance is typically methionine (in eukaryotes) orformylmethionine (prokaryotes). It is also known in the art thateukaryotic and prokaryotic genes may have two or more alternative startcodons, any one of which may be preferentially utilized for translationinitiation in a particular cell type or tissue, or under a particularset of conditions. In the context of the claims, “start codon” and“translation initiation codon” refer to the codon or codons that areused in vivo to initiate translation of an mRNA molecule transcribedfrom a gene encoding human replication-initiation protein, regardless ofthe sequence(s) of such codons. It is also known in the art that atranslation termination codon (or “stop codon”) of a gene may have oneof three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the correspondingDNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively). The terms“start codon region,” “AUG region” and “translation initiation codonregion” refer to a portion of such an mRNA or gene that encompasses fromabout 25 to about 50 contiguous nucleotides in either direction (i.e.,5′ or 3′) from a translation initiation codon. This region is a suitabletarget region. Similarly, the terms “stop codon region” and “translationtermination codon region” refer to a portion of such an mRNA or genethat encompasses from about 25 to about 50 contiguous nucleotides ineither direction (i.e., 5′ or 3′) from a translation termination codon.This region is also a suitable target region. The open reading frame(ORF) or “coding region,” which is known in the art to refer to theregion between the translation initiation codon and the translationtermination codon, is also a region which may be targeted effectively.Other suitable target regions include the 5′ untranslated region(5′UTR), known in the art to refer to the portion of an mRNA in the 5′direction from the translation initiation codon, and thus includingnucleotides between the 5′ cap site and the translation initiation codonof an mRNA or corresponding nucleotides on the gene and the 3′untranslated region (3′UTR), known in the art to refer to the portion ofan mRNA in the 3′ direction from the translation termination codon, andthus including nucleotides between the translation termination codon and3′ end of an mRNA or corresponding nucleotides on the gene. The 5′ capof an mRNA comprises an N7-methylated guanosine residue joined to the5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ capregion of an mRNA is considered to include the 5′ cap structure itselfas well as the first 50 nucleotides adjacent to the cap. The 5′ capregion may also be a suitable target region.

[0027] Although some eukaryotic mRNA transcripts are directlytranslated, many contain one or more regions, known as “introns,” whichare excised from a pre-mRNA transcript to yield one or more mature MRNA.The remaining (and therefore translated) regions are known as “exons”and are spliced together to form a continuous mRNA sequence. mRNA splicesites, i.e., exon-exon or intron-exon junctions, are also suitabletarget regions, and are particularly useful in situations where aberrantsplicing is implicated in disease, or where an overproduction of aparticular mRNA splice product is implicated in disease. Aberrant fusionjunctions due to rearrangements or deletions are also suitable targets.Targeting particular exons in alternatively spliced mRNAs are alsosuitable. Introns are also suitable target regions for antisensecompounds targeted, for example, to DNA or pre-mRNA.

[0028] The term “oligonucleotides” refers to an oligomer or polymer ofnucleotide or nucleoside monomers consisting of naturally occurringbases, sugars, and internucleosidic (backbone) linkages. The term alsoincludes modified or substituted oligomers comprising non-naturallyoccurring monomers or portions thereof, which function similarly. Suchmodified or substituted oligomers may be preferred over naturallyoccurring forms because of the properties such as enhanced cellularuptake, or increased stability in the presence of nucleases. The termalso includes chimeric oligonucleotides which contain two or morechemically distinct regions.

[0029] “Chimeric oligonucleotides” or “chimeras” refer tooligonucleotides that contain two or more chemically distinct regions,each made up of at least one nucleotide. These oligonucleotidestypically contain at least one region of modified nucleotides thatconfers one or more beneficial properties (such as, for example,increased nuclease resistance, increased uptake into cells, increasedbinding affinity for the RNA target) and a region that is a substratefor RNase H cleavage. In one embodiment, a chimeric oligonucleotidescomprises at least one region modified to increase target bindingaffinity, and, usually, a region that acts as a substrate for RNAse H.In one embodiment, at least one nucleotide is modified at the 2′position of the sugar, for example, a 2′-O-alkyl, 2′-O-alkyl-O-alkyl or2′-fluoro-modified nucleotide. Such modifications are routinelyincorporated into oligonucleotides and these oligonucleotides have beenshown to have a higher target binding affinity than2′-deoxyoligonucleotides against a given target. Increased affinitytypically enhances the claimed antisense oligonucleotides inhibition ofhuman replication-initiation protein expression.

[0030] RNAse H is a cellular endonuclease that cleaves the RNA strand ofRNA:DNA duplexes; activation of this enzyme therefore results incleavage of the RNA target, and thus can greatly enhance the efficiencyof antisense inhibition. Cleavage of the RNA target can be routinelydemonstrated by gel electrophoresis. In another embodiment, the chimericoligonucleotides is also modified to enhance nuclease resistance. Cellscontain a variety of exo- and endo-nucleases which can degrade nucleicacids. A number of nucleotide and nucleoside modifications have beenshown to make the oligonucleotides into which they are incorporated moreresistant to nuclease digestion than the native oligodeoxynucleotide.Nuclease resistance may be measured by incubating oligonucleotides withcellular extracts or isolated nuclease solutions and measuring theextent of intact oligonucleotides remaining over time, usually by gelelectrophoresis.

[0031] In one embodiment, the antisense oligonucleotides are ribonucleicor deoxyribonucleic acids and may contain naturally occurring orsynthetic monomeric bases, including adenine, guanine, cytosine, thymineand uracil. As another example, the claimed antisense oligonucleotidesmay also contain modified bases such as 5-methylcytosine (5-me-C orm5c), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further bases include those disclosed in U.S. Pat. No.3,687,808, those disclosed in the Concise Encyclopedia Of PolymerScience And Engineering 1990, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, those disclosed by Englisch et al. (Angewandte Chemie,International Edition 1991, 30, 613-722), and those disclosed bySanghvi, Y. S., Chapter 15, Antisense Research and Applications 1993,pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, and areincorporated by reference herein. Certain of these nucleobases areparticularly useful for increasing the binding affinity of the claimedantisense oligomeric compounds, including 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. Asanother example, 5-methylcytosine substitutions increase nucleic acidduplex stability by 0.61.2.degree. C. Sanghvi, Y. S., Crooke, S. T. andLebleu, B., eds., Antisense Research and Applications 1993, CRC Press,Boca Raton, pages 276-278 and are suitable base substitutions,particularly when combined with 2′-O-methoxyethyl sugar modifications.Modified bases may be prepared, for example, according to U.S. Pat. Nos.3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941,and are incorporated by reference herein. However, other known basemodifications may be used.

[0032] The modifications may also include attachment of other chemicalgroups such as methyl, ethyl, or propyl groups to the various parts ofthe oligonucleotides including the sugar, base or backbone components.Other suitable modified oligonucleotides backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotri-esters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thiono-alkylphosphonates, thionoalkylphosphotriesters, andborano-phosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms may also be used. Modificationscontaining phosphorus-containing linkages may be prepared, for example,according to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; and 5,625,050, which teachings are incorporatedherein by reference. However, other known base modifications may beused.

[0033] Other suitable modified oligonucleotides backbones that may beused do not include a phosphorus atom and have backbones that areformed, for example, by short chain alkyl or cycloalkyl internucleosidelinkages, mixed heteroatom and alkyl or cycloalkyl internucleosidelinkages, or one or more short chain heteroatomic or heterocyclicinternucleoside linkages. These include, for example, those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts. These modifications may be prepared, forexample, according to U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444;5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938;5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225;5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289;5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439,and are incorporated by reference herein. However, other known basemodifications may be used.

[0034] In other embodiments, the antisense oligonucleotides may containphosphorothioate or heteroatom linkages or backbones, for example,CH₂NHOCH₂, CH₂N(CH₃)OCH₂ [also known as a methylene (methylimino) or MMIbackbone], CH₂ON(CH₃)CH₂, CH₂N(CH₃)N(CH₃)CH₂ or ON(CH₃)CH₂CH₂ [whereinthe native phosphodiester backbone is represented as OPOCH₂] of theabove referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove referenced U.S. Pat. No. 5,602,240. Other suitable modificationsinclude phosphorothioate bonds linking between the four to six3′-terminus nucleotides. The phosphorothioate bonds may link all thenucleotides. The phosphorothioate linkages may be mixed R and Senantiomers, or they may be stereoregular or substantially stereoregularin either R or S form. In another embodiment, the oligonucleotides ismodified by at least two phosphorothioate linkages such that eacholigonucleotides contains a phosphorothioate linkage between the firsttwo nucleotides and between the last two nucleotides.

[0035] The claimed antisense oligonucleotides may also contain sugarmimetics. The oligonucleotides may have at least one nucleotide with amodified base and/or sugar, such as a 2′-O-substituted ribonucleotide.The term “2′-O-substituted” means substitution of the 2′ position of thepentose moiety with an —O-lower alkyl group containing 1-6 saturated orunsaturated carbon atoms, or with an —O-aryl or allyl group having 2-6carbon atoms, wherein such alkyl, aryl or allyl group may beunsubstituted or may be substituted, e.g., with halo, hydroxy,trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl,carbalkoxyl, or amino groups. The oligonucleotides of the invention mayinclude four or five ribonucleotides 2′-O-alkylated at their 5′ terminusand/or four or five ribonucleotides 2′-O-alylated at their 3′ terminus.In other embodiments, antisense oligonucleotides may also containmodifications at one of the following at the 2′ position: OH; F; O-, S-,or N-alkyl, O-alkyl-O-alkyl, O, S-, or N-alkenyl, or O-, S- orN-alkynyl, wherein the alkyl, alkenyl and alkynyl may be substituted orunsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl.Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃,O(CH₂)₂. ON(CH₃)₂, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other suitable oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an oligonucleotides, or a group forimproving the pharmacodynamic properties of an oligonucleotides, andother substituents having similar properties. Another suitablemodification includes 2′-methoxyethoxy (2′-O—CH₂CH₂COH₃, also known as2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 78,486-504 (1995)) i.e., an alkoxyalkoxy group. Further preferredmodifications include 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, and 2′-dimethylaminoethoxyethoxy(2′-DMAEOE), and other modifications known in the art.

[0036] For example, other modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotides, for example, at the 31 position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Modifications containing sugar mimetics may be prepared, forexample, according to U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080;5,359,044; 5,393,878; 5,446,137; 5,466,786; 35 5,514,785; 5,519,134;5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053;5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, whichteachings are incorporated herein by reference. However, other knownbase modifications may be used.

[0037] The claimed antisense oligonucleotides may also comprisenucleotide analogues wherein the structure of the nucleotide isfundamentally altered. For example, in peptide nucleic acid (PNA), boththe sugar and the internucleoside linkage, i.e., the backbone, of thenucleotide units are replaced with novel groups. The base units aremaintained for hybridization with an appropriate nucleic acid targetcompound. PNA has been shown to have excellent hybridization properties.In PNA compounds, the sugar-backbone of an oligonucleotides is replacedwith an amide containing backbone, for example, an aminoethylglycinebackbone. The nucleobases are retained and are bound directly orindirectly to aza nitrogen atoms of the amide portion of the backbone.PNA analogues have been shown to be resistant to degradation by enzymesand to have extended lives in vivo and in vitro. PNAs also bind morestrongly to a complementary DNA sequence than to a naturally occurringnucleic acid molecule due to the lack of charge repulsion between thePNA strand and the DNA strand. PNA compounds may be prepared, forexample, according to U.S. Pat. Nos. 5,539,082; 5,714,331; and5,719,262. Further teaching of PNA compounds can be found in Nielsen etal., Science, 254, 1497-1500 (1991), and is incorporated by referenceherein.

[0038] Other embodiments may also include other nucleotides comprisingpolymer backbones, cyclic backbones, or acyclic backbones. For example,suitable nucleotides may comprise morpholino backbone structures (U.S.Pat. No. 5,034,506 (33)) or other modified linkages.

[0039] In other embodiments, the antisense oligonucleotides are“nuclease resistant” when they have either been modified such that theyare not susceptible to degradation by DNA and RNA nucleases oralternatively they have been placed in a delivery vehicle that in itselfprotects the oligonucleotides from DNA or RNA nucleases. Nucleaseresistant oligonucleotides include, for example, methyl phosphonates,phosphorothioates, phosphorodithioates, phosphotriesters, and morpholinooligomers. Suitable delivery vehicles for conferring nuclease resistanceinclude, for example liposomes.

[0040] The claimed antisense oligonucleotides may also contain groupsfor improving the pharmacokinetic properties of an oligonucleotides. Forexample, oligonucleotides may be linked to one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotides. Such moieties include but are notlimited to lipid moieties such as a cholesterol moiety (Letsinger etal., Proc. Natl. Acad. Sci. USA, 86, 6553-6556 (1989)), cholic acid(Manoharan et al., Bioorg. Med. Chem. Lett., 4, 1053-1059 (1994)), athioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad.Sci., 660, 306-309 (1992); Manoharan et al., Bioorg. Med. Chem. Let., 3,2765-2770 (1993)), a thiocholesterol (Oberhauser et al., Nucl. AcidsRes., 20, 533-538 (1992)), an aliphatic chain, e.g., dodecandiol orundecyl residues (Saison-Behmoaras et al., EMBO J., 10, 1111-1118(1991); Kabanov et al., FEBS Lett., 259, 327-330 (1990); Svinarchuk etal., Biochimie, 75, 49-54 (1993)), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 36, 3651-3654 (1995); Shea et al., Nucl. Acids Res.,18, 3777-3783 (1990)), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 14, 969-973 (1995)), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 36,3651-3654 (1995)), a palmityl moiety (Mishra et al., Biochim. Biophys.Acta, 1264, 229-237 (1995)), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 277, 923-937 (1996)). However, otherpharmacokinetic-enhancing moieties known in the art are also suitable.For example, many of these conjugates may be prepared according to U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, andare incorporated by reference herein. However, other knownpharmacokinetic-enhancing modifications may be used.

[0041] In other embodiments, antisense oligonucleotides includemutations, such as substitutions, insertions and deletions. Preferably,there will be less that 10% of the sequence having mutations. In oneembodiment, the antisense oligonucleotides sequence is at least 90%complementary to the target sequence.

[0042] It is not necessary for all positions in a given compound to beuniformly modified, and in fact, more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotides.

[0043] The claimed antisense oligonucleotides may be formed by solidphase synthesis or any other means known in the art. Equipment for suchsynthesis is sold by several vendors including Applied Biosystems.Similar techniques to prepare modified oligonucleotides such asphosphorothioates and alkylated derivatives are also known, as aretechniques and commercially available modified amidites andcontrolled-pore glass (CPG) products such as biotin, fluorescein,acridine or psoralen-modified amidites and/or CPG (available from GlenResearch, Sterling Va.) to synthesize fluorescently labeled,biotinylated or other modified oligonucleotides such ascholesterol-modified oligonucleotides.

[0044] In one embodiment, antisense oligonucleotides are selected fromthe sequence complementary to the human replication-initiation gene suchthat the sequence exhibits little likelihood of showing duplexformation, hair-pin formation, and homooligomer/sequence repeats, buthas an increased potential to bind to the replication-initiation genesequences. These properties may be determined using a suitable computermodeling program such as Mfold. Zuker, M., In “RNA Biochemistry andBiotechnology”, J. Barciszewski & B. F. C. Clark, eds., NATO ASI Series,Kluwer Academic Publishers, incorporated by reference herein.

[0045] In one embodiment, antisense oligonucleotides generally comprisefrom at least about 3 nucleotides or nucleotide analogs, more preferablythey are at least about 5 nucleotides, more preferably they are at leastabout 7 nucleotides, more preferably they are at least about 9nucleotides, and most preferably they are at least about 16 nucleotides.The antisense oligonucleotides are preferably less than about 100nucleotides or nucleotide analogs, more preferably, less than about 50nucleotides or nucleotide analogs, most preferably less than about 35nucleotide or nucleotide analogs. In one embodiment, the antisensoligonucleotides has the sequence identified in SEQ. ID. Nos. 1, 4,7,10, 13, 16, 19, 22, or 25. In another embodiment, the sequencecontains an 8 nucleotide base portion selected from SEQ. ID. Nos. 1, 4,7, 10, 13, 16, 19 22, and 25.

[0046] Other embodiments of antisense oligonucleotides includebioequivalent compounds, for example, pharmaceutically acceptable saltsand prodrugs, such as salts, esters, or salts of such esters, or anyother compound which, upon administration to an animal including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. For example, embodiments includeacceptable salts of the nucleic acids of oligonucleotides and prodrugsof such nucleic acids. Pharmaceutically acceptable salts arephysiologically and pharmaceutically acceptable salts of the nucleicacids of the invention: i.e., salts that retain the desired biologicalactivity of the parent compound and do not impart undesiredtoxicological effects thereto (see, for example, Berge et al.,“Pharmaceutical Salts,” J. of Pharma Sci., 66, 1-19 (1977)),incorporated by reference herein.

[0047] Suitable pharmaceutically acceptable salts include but are notlimited to (a) salts formed with cations such as sodium, potassium,ammonium, magnesium, calcium, polyamines such as spermine andspermidine, etc.; (b) acid addition salts formed with inorganic acids,for example hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, nitric acid and the like; (c) salts formed with organicacids such as, for example, acetic acid, oxalic acid, tartaric acid,succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid,malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid,alginic acid, polyglutamic acid, naphthalenesulfonic acid,methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonicacid, polygalacturonic acid, and the like; and (d) salts formed fromelemental anions such as chlorine, bromine, and iodine.

[0048] In another embodiment, the antisense oligonucleotides mayadditionally or alternatively be prepared to be delivered in a Aprodrug™form. The term Aprodrug™ refers to a therapeutic agent that is preparedin an inactive form that is converted to an active form (i.e., drug)within the body or cells thereof by the action of endogenous enzymes orother chemicals and/or conditions. In one embodiment, prodrug versionsof claimed oligonucleotides are prepared as SATE [(S-acetyl-2-thioethyl)phosphate] derivatives according to the methods disclosed in WO 93/24510to Gosselin et al., published Dec. 9, 1993, incorporated by referenceherein.

[0049] In another aspect, methods for inhibiting humanreplication-initiation protein expression and for interfering with cellhyperproliferation were developed using antisense oligonucleotidestargeted to portions of human replication-initiation mRNA. In oneembodiment, a method of treating or preventing a hyperproliferativecondition comprises administering to a human or cells thereof atherapeutically effective amount of antisense oligonucleotides having asequence complementary to a target sequence encoding mRNA of a humanreplication-initiation gene, wherein expression of thereplication-initiation protein is inhibited. In another embodiment, thehyperproliferating cells are contacted with a therapeutically effectiveamount of claimed antisense oligonucleotides. In another embodiment,tissues or cells are contacted with oligonucleotides. To “contact”tissues or cells with an oligonucleotides or oligonucleotides means toadd the oligonucleotides, for example, in a liquid carrier, to a cellsuspension or tissue sample, either in vitro or ex vivo, or toadminister the oligonucleotides to cells or tissues within an animal.However, other contact means known in the art may be used. It will beappreciated by those skilled in the art that equivalent methods oragents that inhibit the expression and/or activities ofreplication-initiation proteins are contemplated, for example, RNAi, andincluding compounds such siRNA or the like in RNA Interference forinhibition of gene expression as described in Harboth et al.,“Identification of essential genes in cultured mammalian cells usingsmall interfering RNAs” Journal of Cell Science 114, 4557-4565 (2001),which teachings are incorporated herein by reference.

[0050] In one embodiment, the hyperproliferative condition is cancer,angiogenesis, neovascularization, psoriasis, blood vessel restenosis, oratherosclerosis or similar condition. In another embodiment, thetargeted sequence encodes a portion of hCdc6, hCdc45, hMcm2, hMcm3,hMcm4, hMcm5, hMcm6, hMcm7, hOrc1, hOrc2, hOrc3, hOrc4, hOrc5, hOrc6 orhCdt1 genes.

[0051] claimed antisense oligonucleotide compounds may be formulated ina pharmaceutical composition, which may include pharmaceuticallyacceptable carriers, thickeners, diluents, buffers, preservatives,surface active agents, neutral or cationic lipids, lipid complexes,liposomes, penetration enhancers, carrier compounds and otherpharmaceutically acceptable carriers or excipients and the like inaddition to the oligonucleotides. Such compositions and formulationsreadily known in the art and easily incorporated with embodiment of thepresent claims.

[0052] Pharmaceutical compositions comprising the claimed antisenseoligonucleotides of the present invention may also include penetrationenhancers in order to enhance the alimentary delivery of theoligonucleotides. Penetration enhancers may be classified as belongingto one of five broad categories, i.e., fatty acids, bile salts,chelating agents, surfactants and non-surfactants (Lee et al., CriticalReviews in Therapeutic Drug Carrier Systems, 8, 91-192 (1991);Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 7, 1-33(1990)), incorporated by reference herein. One or more penetrationenhancers from one or more of these broad categories may be included.

[0053] Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid, myristic acid, palmitic acid, stearic acid, `linoleic acid,linolenic acid, dicaprate, tricaprate, recinleate, monoolein (a.k.a.1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, mono- and di-glycerides and physiologically acceptablesalts thereof (i.e., oleate, laurate, caprate, myristate, palmitate,stearate, linoleate, etc.) (Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, p. 92 (1991); Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 7, 1 (1990); El-Hariri et al., J.Pharm. Pharmacol., 44, 651-654 (1992)), incorporated by referenceherein. Complex formulations comprising one or more penetrationenhancers may be used. For example, bile salts may be used incombination with fatty acids to make complex formulations.

[0054] The physiological roles of bile include the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 In: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill, New York,N.Y., 1996, pages 934-935), incorporated by reference herein. Variousnatural bile salts, and their synthetic derivatives, act as penetrationenhancers. Thus, the term “bile salt” includes any of the naturallyoccurring components of bile as well as any of their syntheticderivatives.

[0055] Chelating agents include, but are not limited to, disodiumethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g.,sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines) (Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, p. 92 (1991); Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 7, 1-33 (1990); Buur et al., J.Control Rel., 14, 43-51 (1990)), incorporated by reference herein.Chelating agents have the added advantage of also serving as DNaseinhibitors.

[0056] Surfactants include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, p. 92(1991)); and perfluorochemical emulsions, such as FC-43 (Takahashi etal., J. Pharm. Phamacol., 40, 252-257 (1988)), incorporated by referenceherein.

[0057] Non-surfactants include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, p. 92 (1991)),incorporated by reference herein; and non-steroidal anti-inflammatoryagents such as diclofenac sodium, indomethacin and phenylbutazone(Yamashita et al., J. Pharm. Pharmacol., 39, 621-626 (1987)), andincorporated by reference herein.

[0058] As used herein, “carrier compound” refers to a nucleic acid, oranalog thereof, which is inert (i.e., does not possess biologicalactivity per se) but is recognized as a nucleic acid by in vivoprocesses that reduce the bioavailability of a nucleic acid havingbiological activity by, for example, degrading the biologically activenucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically withan excess of the latter substance, can result in a substantial reductionof the amount of nucleic acid recovered in the liver, kidney or otherextracirculatory reservoirs, presumably due to competition between thecarrier compound and the nucleic acid for a common receptor.

[0059] In contrast to a carrier compound, a “pharmaceutically acceptablecarrier” (excipient) is a pharmaceutically acceptable solvent,suspending agent or any other pharmacologically inert vehicle fordelivering one or more nucleic acids to an animal. The pharmaceuticallyacceptable carrier may be liquid or solid and is selected with theplanned manner of administration in mind so as to provide for thedesired bulk, consistency, etc., when combined with a nucleic acid andthe other components of a given pharmaceutical composition. Typicalpharmaceutically acceptable carriers include, but are not limited to,binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose andother sugars, microcrystalline cellulose, pectin, gelatin, calciumsulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate,etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidalsilicon dioxide, stearic acid, metallic stearates, hydrogenatedvegetable oils, corn starch, polyethylene glycols, sodium benzoate,sodium acetate, etc.); disintegrates (e.g., starch, sodium starchglycolate, etc.); or wetting agents (e.g., sodium lauryl sulphate,etc.). Sustained release oral delivery systems and/or enteric coatingsfor orally administered dosage forms are described in U.S. Pat. Nos.4,704,295; 4,556,552; 4,309,406; and 4,309,404, incorporated byreference herein. However, other pharmaceutically acceptable carriersknown in the art may be used.

[0060] The claimed compositions of the present invention mayadditionally contain other adjunct components conventionally found inpharmaceutical compositions at their art-established usage levels. Forexample, the compositions may contain additional compatiblepharmaceutically-active materials such as, e.g., antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the composition of present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of theclaimed compositions.

[0061] The formulation of claimed compositions and their subsequentadministration is believed to be within the skill in the art. Ingeneral, for therapeutics, a patient suspected of needing such therapyis given an oligonucleotides in accordance with the invention, commonlyin a pharmaceutically acceptable carrier, in amounts and for periodswhich will vary depending upon the nature of the particular disease, itsseverity and the patient's overall condition.

[0062] Suitable pharmaceutical compositions may be administered in anumber of ways depending upon whether local or systemic treatment isdesired and upon the area to be treated. Administration may be topical(including but not limited to ophthalmic, vaginal, rectal, intranasal,epidermal, and transdermal), oral or parenteral. Parenteraladministration may include, for example, intravenous drip, intravenousinjection, subcutaneous, intraperitoneal, intraocular, intravitreal orintramuscular injection, pulmonary administration, e.g., by inhalationor insufflation, or intracranial, e.g., intrathecal or intraventricular,administration.

[0063] Formulations for topical administration may include transdermalpatches, ointments, lotions, creams, gels, drops, suppositories, sprays,liquids and powders. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be necessary ordesirable. However, other formulations known in the art may also beused.

[0064] Compositions for oral administration include, for example,powders or granules, suspensions or solutions in water or non-aqueousmedia, capsules, sachets, or tablets. Thickeners, flavorings, diluents,emulsifiers, dispersing aids or binders may be desirable. Practitionersin the art understand that other oral compositions known in the art mayalso be used.

[0065] Compositions for parenteral administration may include sterileaqueous solutions that may also contain buffers, diluents and othersuitable additives. In some cases it may be more effective to treat apatient with claimed oligonucleotides in conjunction with othertraditional therapeutic modalities in order to increase the efficacy ofa treatment regimen. The term “treatment regimen” refers to therapeutic,palliative and prophylactic modalities. For example, a patient may betreated with conventional chemotherapeutic agents, particularly thoseused for tumor and cancer treatment. In another embodiment, the claimedcomposition comprises a chemotherapeutic agent and a claimed antisenseoligonucleotides that inhibits expression of humanreplication-initiation protein in cells. Examples of suchchemotherapeutic agents include but are not limited to daunorubicin,daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin,esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D,mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen,dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine,mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,6-thioguanine, cytarabine (CA), 5-azacytidine, hydroxyurea,deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil(5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine,taxol, vincristine, vinblastine, etoposide, trimetrexate, teniposide,carboplatin, topotecan, irinotecan, gemcitabine, cisplatin anddiethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosisand Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al., eds., Rahway,N.J., incorporated by reference herein. Obviously, otherchemotherapeutic agents known in the art may be used. When used withclaimed antisense oligonucleotides, such chemotherapeutic agents may beused individually (e.g., 5-FU and oligonucleotides), sequentially (e.g.,5-FU and oligonucleotides for a period of time followed by MTX andoligonucleotides), or in combination with one or more other suchchemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotides, or 5-FU,radiotherapy and oligonucleotides).

[0066] Other drugs such as leucovorin, which is a form of folic acidused as a “rescue” after high doses of methotrexate or other folic acidagonists, may also be administered. In some embodiments, 5-FU andleucovorin are given in combination as an IV bolus with the compounds ofthe invention being provided as an IV infusion.

[0067] In addition to such pharmaceutical carriers, cationic lipids maybe included in the formulation to facilitate oligonucleotides uptake.One such composition shown to facilitate uptake is Lipofectin (BRL,Bethesda Md.).

[0068] Regardless of the method by which the claimed antisenseoligonucleotides of the invention are introduced into a patient,colloidal dispersion systems may be used as delivery vehicles to enhancethe in vivo stability of the oligonucleotides and/or to target theoligonucleotides to a particular organ, tissue or cell type. Colloidaldispersion systems include, but are not limited to, macromoleculecomplexes, nanocapsules, microspheres, beads and lipid-based systemsincluding oil-in-water emulsions, micelles, mixed micelles, liposomesand lipid:oligonucleotides complexes of uncharacterized structure. Apreferred colloidal dispersion system is a plurality of liposomes.Liposomes are microscopic spheres having an aqueous core surrounded byone or more outer layers made up of lipids arranged in a bilayerconfiguration (see, generally, Chonn et al., Current Op. Biotech., 6,698-708 (1995)), incorporated by reference herein.

[0069] Dosing of the claimed antisense oligonucleotides is dependent onseverity and responsiveness of the condition to be treated, with courseof treatment lasting from several days to several months or until a cureis effected or a diminution of disease state is achieved. Optimal dosingschedules can be calculated from measurements of drug accumulation inthe body. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual oligonucleotides,and can generally be calculated based on EC50's in in vitro and in vivoanimal studies. For example, given the molecular weight of compound(derived from oligonucleotides sequence and chemical structure) and aneffective dose such as an IC50, for example (derived experimentally), adose in mg/kg is routinely calculated.

[0070] One embodiment diagnoses abnormal proliferative states in tissueor other samples from patients, for example, suspected of having ahyperproliferative disease such as cancer, angiogenesis,neovascularization, psoriasis, blood vessel restenosis, atherosclerosis.However, other abnormal proliferative conditions are contemplated withinthe scope of the claims. For example, in one embodiment, an effectiveamount of claimed antisense oligonucleotides is administered to orcontacted with human cells wherein the antisense oligonucleotides has asequence complementary to a target sequence encoding mRNA of a humanreplication-initiation gene, wherein expression of thereplication-initiation protein is inhibited. An effective amount ofclaimed oligonucleotides is readily determined by the skilledpractitioner.

[0071] A number of assays may be formulated employing the claimedantisense oligonucleotides. For example, one assay comprises contactinga tissue sample with an oligonucleotides of the invention underconditions selected to permit detection and, usually, quantitation ofsuch inhibition. In another embodiment, an assay using the claimedantisense oligonucleotides distinguish tumors associated with humanreplication-initiation protein from tumors having other etiologies, inorder that an efficacious treatment regime can be designed.

[0072] In other embodiments, the claimed methods and antisenseoligonucleotides are used in diagnostics, therapeutics, prophylaxis, andas research reagents and in kits. Since the claimed antisenseoligonucleotides hybridize to nucleic acids encoding a humanreplication-initiation protein, sandwich, colorimetric and other assaysare easily be constructed to exploit this fact. Detection andquantitation of oligonucleotides assays are skills readily known in theart. For example, enzyme conjugation, radiolabelling or any othersuitable detection systems may be used to detect hybridization of theclaimed oligonucleotides. Kits for detecting the presence or absence ofhuman replication-initiation protein may also be prepared.

[0073] The claimed methods and antisense oligonucleotides may also beused for research purposes. Thus, the specific hybridization exhibitedby the oligonucleotides may be used for assays, purifications, cellularproduct preparations and in other methodologies which may be appreciatedby persons of ordinary skill in the art.

[0074] The claimed antisense oligonucleotides are also useful fordetection and diagnosis of human replication-initiation proteinexpression. For example, radiolabeled oligonucleotides can be preparedby ³²P labeling at the 5′ end with polynucleotide kinase (Sambrook etal., Molecular Cloning. A Laboratory Manual, Cold Spring HarborLaboratory Press, 1989, Volume 2, p. 10.59), incorporated by referenceherein. Radiolabeled oligonucleotides are then contacted with tissue orcell samples suspected of human replication initation protein expressionand the sample is washed to remove unbound oligonucleotides.Radioactivity remaining in the sample indicates bound oligonucleotides(which in turn indicates the presence of human replication-initiationprotein) and can be quantitated, for example, using a scintillationcounter or other means readily known in the art. Radiolabeledoligonucleotides can also be used to perform autoradiography of tissuesto determine the localization, distribution and quantitation of humanreplication inititation protein expression for research, diagnostic ortherapeutic purposes. In these embodiments, tissue sections are treatedwith radiolabeled oligonucleotides and washed as described above, thenexposed to photographic emulsion according to routine autoradiographyprocedures. The emulsion, when developed, yields an image of silvergrains over the regions expressing human replication-initiation protein.Quantitation of the silver grains permits protein expression to bedetected.

[0075] Analogous assays for fluorescent detection of humanreplication-initiation expression employ claimed antisenseoligonucleotides that are conjugated with fluorescein or otherfluorescent tag instead of radiolabeling. Such conjugations areroutinely accomplished during solid phase synthesis using fluorescentlylabeled amidites or CPG (e.g., fluorescein-labeled amidites and CPGavailable from Glen Research, Sterling Va. See 1993 Catalog of Productsfor DNA Research, Glen Research, Sterling Va., p. 21), incorporated byreference herein.

[0076] Each of these assay formats is known in the art and is readilyadapted as methods for detection of human replication-initiation proteinexpression in accordance with the claims as a novel and useful means todetect human replication-initiation protein expression.

[0077] Another aspect provides a method of identifying antisenseoligonucleotides that inhibit expression of a gene. In one embodiment,the method involves screening an antisense oligonucleotides forinhibition of gene expression, wherein the oligonucleotides contains aphosphodiester DNA backbone. After selecting one or more antisenseoligonucleotides from the screening that inhibited gene expression, atleast two internucleosidic linkages of the selected antisenseoligonucleotides are modified such that the oligonucleotides contains aphosphorothioate linkage between the first two nucleotides and betweenthe last two nucleotides of the sequence. Screening the modifiedoligonucleotides for inhibition of gene expression results in one ormore antisense oligonucleotides that may be selected that activelyinhibit gene expression as well as have partially modified backbones.Once these active antisense oligonucleotides have been selected, one ormore linkages of the oligonucleotides obtained from the second selectingstep is replaced with more phosphorothioate linkages.

[0078] For example, in one embodiment, antisense oligonucleotides weredesigned, screened and tested according to the present claims. Extensivebase pairing exists in the secondary structure of mRNAs, therefore, onlythe single-stranded regions of mRNAs are potential targets for antisenseoligonucleotides. With the known sequences of the cDNA of thereplication-initiation proteins in human (Williams, U.S. Pat. No.5,851,821; Saha, P. et al., J. Biol. Chem., 50, 6075-6086 (1990);Todorov, I. T, et al., J. Cell Sci., 107, 253-265 (1994)), incorporatedby reference herein, computer modeling, for example Mfold (Zuker, M., In“RNA Biochemistry and Biotechnology”, J. Barciszewski & B.F.C. Clark,eds., NATO ASI Series, Kluwer Academic Publishers.) was used to predictthe secondary structures of mRNAs, and then antisense oligonucleotideswere targeted to the putative single-stranded areas along the entirelength of the mRNAs. One hundred and seventy antisense oligonucleotideswith normal phosphodiester DNA backbones targeted to the hCdc6, hMcm2and hCdc45 genes were designed and subjected to initial screening fortheir activities to inhibit cancer cell growth in tissue culture. Of the170 oligonucleotides, 66 were targeted to hCdc6 (named hC6-1 throughhC6-66), 64 to hMcm2 (hM2-1 through hM2-64), and 40 to hCdc45 (hC45-1through hC45-40). All 170 oligonucleotides and the modifiedoligonucleotides containing phosphorothioate linkages described belowwere custom-synthesized and purified to ˜99% (HPSF™—“Highly PurifiedSalt Free”—grade; MGW Biotech), and were further ethanol-precipitatedtwice to remove small-molecule impurities.

[0079] The human cancer cell lines initially used were a liver cancercell line (called the Chang's Liver Cancer Cells) and Hela cells. Otherhuman cancer (such as HoNel, T-Tn, HepG2 and Hep3B) and normal (L-02)cell lines were also used to test the activities and specificity of theoligonucleotides. Cells (5000/well) were seeded in 96-well plates andgrown in 100 μl/well of the DMEM medium (Life Technologies) containingfetal bovine serum (10%), penicillin and streptomycin. Cells wereincubated at 37° C. with 5% CO₂. One day later, the medium was changedto the same medium (50 μl) but without serum, and the oligonucleotides(1 μM) were added to the cell culture as conjugates with the cationicliposome carrier Lipofectin (1.7%) or LipofectAMiNE Plus (2.4%) (LifeTechnologies) in a total transfection volume of 70 μl. A mixture of all170 oligonucleotides (1 μM total) conjugated with the carrier, thecarrier without oligo, and individual oligonucleotides without thecarrier were used as the negative controls. Three hours later, themedium was changed back to the regular medium containing serum. Two dayslater, the number of viable cells was determined by the tetrazoliumassay using WST-1 as described (Ishiyama et al., 1996). The WTS-1 assayis more sensitive and accurate and has a wider dynamic range than theMTT assay. It was found that 16 of the 170 oligonucleotides tested couldinhibit cancer cell growth and kill cancer cells, resulting in thenumber of viable cells being 20-40% compared to the untreated cells.Oligonucleotides without the carrier had no activity (90-100% of cellssurvived), while the carrier without oligo or the mixture of all 170oligonucleotides conjugated with the carrier gave 60-70% live cellscompared to the untreated cells. These results indicated that while thecarrier was necessary, it was somewhat cytotoxic when used in much ahigh concentration that was needed to carry the large amount ofoligonucleotides with phosphodiester DNA backbones into the cells. (Thetoxicity of the carrier was reduced by lowering the concentrations ofthe carriers for the modified oligonucleotides in later experiments;below.)

[0080] The 16 antisense oligonucleotides that were tested active in theinitial screening were then subjected to further testing for theiractivities towards the cancer cells. To increase resistance toexonucleases which are present in the growth medium containing serum,both the 5′- and 3′- ends of the oligonucleotides were modified byphosphorothioate (PT) linkages, such that each oligo contains a PTlinkage between the first two nucleotides and between the last twonucleotides. The sense and mismatched oligonucleotides that werePT-modified in the same way were used as the negative controls. Notethat the sense oligonucleotides are complementary to the correspondingantisense oligonucleotides and that 3 to 4 of the nucleotides in eachantisense oligo were exchanged to generate the mismatched oligo, keepingbase composition unchanged (Table 1).

[0081] While the cells were grown in the same way as described above,the end-modified oligonucleotides (0.7 μM) were transfected into thecells as conjugates with LipofectAMINE Plus (2%) or LipofectAMINE-2000(0.7%) in 70 μl or 100 μl, respectively, of the Opti-MEM medium (LifeTechnologies) without serum as the transfection medium. Three (forLipofectAMINE Plus) or four (for LipofectAMINE-2000) hours later, themedium was changed back to the regular growth medium containing serum.During screening of these modified oligonucleotides, the number ofviable cells was determined by the WST-1 assay two dayspost-transfection. Nine of these end-modified oligonucleotides showedstrong activities to not only prevent cancer cell growth, but also toinduce cancer cell death in culture. Two days after a single treatment,the number of live cancer cells was ranged from 6.7% (with hC45-18a) to18.9% (with hC45-30a) compared to the untreated cells (Table 1).Moreover, actual cell death and cell lysis were evident as observedunder a light microscope and demonstrated by a series of cell deathassays, for example, measuring DNA fragmentation on the activity of LDHdehydrogenase released into the medium upon cell death. One or both ofthe negative control oligonucleotides for five of the nine activeantisense oligonucleotides (hC6-35, hM2-47, hC45-18, hC45-27 andhC45-30) were not nearly as active as the antisense oligonucleotides,indicating good specificity of these four antisense oligonucleotidestowards the targeted genes. However, the sense and mismatched controloligonucleotides for the other four active antisense oligonucleotides(hC6-39, hC6-60, hM2-13 and hM2-34) were also quite active. Theactivities of the control oligonucleotides were likely due to unintendedinhibition of other unknown genes or cell functions by these controloligonucleotides, which does not necessarily mean that the antisenseoligonucleotides were non-specific in their anti-cancer cell activities.TABLE 1 Summary of the Nine End-Modified Antisense Oligonucleotides withAnti-Cancer Cell Activities SEQ. % Oligo Length ID Viable Name^(a) (nt)No. Oligo Sequence (5′→3′)^(c) Cells^(d) hC6- 16  1 AAG GTG GGA AGT TCAA 13.5 35a hC6- 16  2 AAG aTG GGt AGg TCA A 31.5 35m hC6- 16  3 TTG AACTTC CCA CCT T 28.1 35s hC6- 18  4 CTC CCT CTT GGC TCA AGG 15.3 39a hC6-18  5 CTC CCa CCT GGt TCt AGG 7.3 39m hC6- 18  6 CCT TGA GCC AAG AGG GAG21.8 39s hC6- 19  7 AGC CTG GCC AAC ATG GTA A 9.1 60a hC6- 19  8 AGC CgGaCC AgC ATt GTA A 18.1 60m hC6- 19  9 TTA CCA TGT TGG CCA GGC T 16.1 60shM2- 16 10 CTT GAA GAC GTT GTG G 13.7 13a hM2- 16 11 CTT tAA GgC GTa GTGG 19.4 13m hM2- 16 12 CCA CAA CGT CTT CAA G 21.1 13s hM2- 16 13 CAG AACGAG GGC CCC A 9.9 34a hM2- 16 14 GAG cAG GAG GcC aCC A 11.1 34m hM2- 1615 TGG GGC CCT GGT TCT G 9.9 34s hM2- 17 16 TCC CGC AGA TGG ATG CG 18.947a hM2- 17 17 TCC CtC AGg TGG AaG CC 63.3 47m hM2- 17 18 CGC ATC CATCTG CGG GA 34.1 47s hC45- 20 19 AGG CTG TCA TGG AGG GAC CA 6.7 18a hC45-20 20 AGG CTc TgA gGG AGt GAG CA 9.5 18m hC45- 20 21 TGG TCC CTC CAT GACAGC CT 39.1 18s hC45- 19 22 CCC GCA TGT CCT TCA TCC C 11.8 27a hC45- 1923 CGC GtA TGc CCa TCt TCC C 30.7 27m hC45- 19 24 GGG ATG AAG GAC ATGCGC G 12.1 27s hC45- 16 25 GAA GTG ATC TGT CCC T 18.4 30a hC45- 16 26GAg GTG AaC TtT CCC T 50.1 30m hC45- 16 27 AGG GAG AGA TCA CTT C 55.630s

[0082] The nine active antisense oligonucleotides were checked to see ifthey actually inhibited the expression of the targeted genes. Total RNAwas extracted using the TRIzol kit (Life Technologies) according to themanufacturer's instruction, and proteins were extracted using a commonlyused lysis buffer (50 mM HEPES, pH 7.6, 150 mM NaCl, 1 mM EGTA, 1.5 mMMgCl₂, 10% glycerol, 2 mM DTT, 1% Triton X-100, plus protease andphosphatase inhibitors). The mRNA levels were examined by RT-PCR usingspecific primers to the targeted and control genes and the proteins byWestern blotting using specific antibodies to the proteins encoding bythe targeted genes. It was found that the antisense oligonucleotidesthat were active in preventing cancer cell growth and killing cancercells reduced the levels of both the mRNAs and proteins of the targetedgenes by 70-90% compared to the untreated cells and those treated withcontrol oligonucleotides or carrier alone (see Examples below).

[0083] Four of the active and relatively specific antisenseoligonucleotides (hC6-35a, hM247a, hC45-18a, and hC45-30a) were chosenfor further characterization with the oligonucleotides fully modifiedwith phosphorothioate (PT) linkages (resistant to both exo- andendo-nuclease digestion). The four fully PT-modified antisenseoligonucleotides were active in much lower concentrations (at 0.1-0.2μM) than the phosphodiester (1 μM) or end-modified oligonucleotides (0.7μM) with the same DNA sequences.

[0084] To assist in understanding the present invention, the followingexamples are included and describe the results of a series ofexperiments. The following examples relating to this invention shouldnot be construed to specifically limit the invention or such variationsof the invention, now known or later developed, which fall within thescope of the invention as described and claimed herein.

EXAMPLES Example 1

[0085] Initial Screening of the 170 Antisense Oligonucleotides withPhosphodiester DNA Backbones. The Chang's Liver Cancer Cells and Helacells were used to test the activities and specificity of theoligonucleotides. Cells (5000/well) were seeded in 96-well plates andgrown in 100 μl/well of DMEM medium containing fetal bovine serum (10%),penicillin and streptomycin. One day later, the medium was changed tothe same medium (50 μl) but without serum, and the oligonucleotides (1μM) were added to the cell cultures as conjugates with Lipofectin (1.7%)or LipofectAMINE Plus (2.4%) (The total transfection volume was 70 μl).A mixture of all 170 oligonucleotides (1 μM total) conjugated with thecarrier, the carrier without oligo, and individual oligonucleotideswithout the carrier were used as the negative controls. Three hourslater, the medium was changed back to the regular medium containingserum. Two days later, the number of viable cells was determined by theWST-1 assay. Among the 170 oligonucleotides tested, 16 of them couldinhibit cell growth and kill cancer cells; 20-40% of cells remainedviable compared to the untreated cells. Oligonucleotides without thecarrier had no activity (90-100% of cells survived), while the carrierwithout oligo or the mixture of all oligonucleotides conjugated with thecarrier resulted in 60-70% live cells compared to the untreated cells.The carrier was somewhat cytotoxic when used in much a highconcentration that was needed for carrying the large amounts ofoligonucleotides with phosphodiester DNA backbones into the cells. (Thetoxicity of the carrier was minimized by lowering the concentrations ofthe carriers used for the modified oligonucleotides in laterexperiments; below.)

Example 2

[0086] Further Testing of the 16 Oligonucleotides That ShowedAnti-Cancer Cell Activities in the Initial Screening. The 16 antisenseoligonucleotides and their corresponding mismatched and sense controloligonucleotides were produced with their 5′- and 3′-end modified withphosphorothioate linkages, and tested for their activities towardscancer cells. The oligonucleotides (0.7 μM) were transfected into theChang's Liver Cancer Cells or Hela cells as conjugates withLipofectAMINE Plus (2%) or LipofectAMINE-2000 (0.7%) in 70 μl or 100 μl,respectively, of the Opti-MEM medium without serum as the transfectionmedium. The number of viable cells was measured as described in Example1 and cell death was measured by the LDH dehydrogenase release assay.Nine of these 16 oligonucleotides showed strong anti-cancer cellactivities (Table 1). The number of viable cancer cells in theoligo-treated culture was ranged from 6.7% (for hC45-18a) to 18.9% (forhC45-30a) compared to the untreated cells. One or both of the negativecontrol oligonucleotides for five antisense oligonucleotides (hC6-35,hM2-47, hC45-18, hC45-27 and hC45-30) were not nearly as active as theantisense oligonucleotides. However, the sense and mismatched controloligonucleotides for the other four antisense oligonucleotides (hC6-39,hC6-60, hM213 and hM2-34) were also quite active, perhaps due tounintended inhibition of other unknown genes or cell functions by thesecontrol oligonucleotides.

Example 3

[0087] The Antisense Oligonucleotides hM2-47a with Anti-Cancer CellActivities Inhibited the Expression of the Target Gene hMcm2, But Not ofhCdc6 or hCdc45. The Chang's Liver Cancer Cells were treated with theantisense oligonucleotides hM247a (0.7 μM) or the correspondingmismatched (hM247m) or sense (hM2-47s) control oligonucleotides (seeTable 1) conjugated with LipofectAMINE-2000 (0.7%) in 100 μl of theOpti-MEM medium without serum for 4 hrs. The cells were then grown inregular DMEM containing serum for 1.5 hrs before RT-PCR analysis wascarried out with total RNA isolated from the treated and untreated cellsusing the TRIzol Reagent (Life Technologies). RNA wasreverse-transcribed into first strand cDNA using a cDNA synthesis kit(MBI Fermentas) with oligo(dT) as the primer. The same cDNA sample wasthen used as the template for PCR amplification of a specific fragmentof hMcm2, hCdc6, and hCdc45, respectively, while a fragment of theinternal control β-actin gene was co-amplified with each of the threeinitiation genes (FIG. 1). The antisense oligonucleotides hM2-47aspecifically reduced the mRNA level of the target gene hMcm2 (Lane 3),but not the other replication-initiation gene hCdc6 (Lanes 8) or hCdc45(Lane 13). UT: untreated cells; LP: liposome without oligonucleotides;A: antisense oligonucleotides; M: mismatched oligonucleotides.

Example 4

[0088] The Antisense Oligonucleotides hC45-30a with Anti-Cancer CellActivities Inhibited the Expression of the Target Gene hCdc45. But Notof hMcm2 or hCdc6. The Chang's Liver Cancer Cells were treated with theantisense (A) oligonucleotides hC45-30a (0.7 μM) or the correspondingmismatched (M) or sense (S) control oligonucleotides (see Table 1) inthe same way as described in Example 3. The levels of the mRNA ofhCdc45, hCdc6, hMcm2, and the β-actin gene, respectively, were measuredby RC-PCR analysis (FIG. 2) as described in Example 3. The antisenseoligonucleotides hC45-30a specifically diminished the mRNA level of thetarget gene hCdc45 (Lane 3), but not the other replication-initiationgene hCdc6 (Lanes 8) or hMcm2 (Lane 13). UT: untreated cells; LP:liposome without oligonucleotides.

Example 5

[0089] The Antisense Oligonucleotides hC6-35a with Anti-Cancer CellActivities Reduced the Expression of the Target Gene hCdc6. But Not ofhMcm2 or hCdc45. The Chang's Liver Cancer Cells were treated with theantisense (A) oligonucleotides hC6-35a (0.7 μM) or the correspondingmismatched (M) or sense (S) control oligonucleotides (see Table 1) inthe same way as described in Example 3. The levels of the mRNA of hCdc6,hMcm2, hCdc45, and the β-actin gene, respectively, were measured byRC-PCR analysis (FIG. 3) as described in Example 3. The antisenseoligonucleotides hC6-35a specifically reduced the mRNA level of thetarget gene hCdc6 (Lane 3), but not the other replication-initiationgene hMcm2 (Lanes 8) or hCdc45 (Lane 13). UT: untreated cells; LP:liposome without oligonucleotides.

Example 6

[0090] The Antisense Oligonucleotides That Are Targeted to hCdc6 andHave Anti-Cancer Cell Activities Reduced the hCdc6 Protein Level. TheChang's Liver Cancer Cells were treated with the antisenseoligonucleotides hC6-35a, hC6-39a, or hC6-60a (0.7 μM) (see Table 1)conjugated with LipofectAMINE-2000 (1%) in 100 μl of the Opti-MEM mediumwithout serum for 4 hrs. The corresponding mismatched oligonucleotideswere used as the negative control. The cells were then grown in regularDMEM containing serum for 1.5 hrs before proteins were extracted andWestern blotted with a monoclonal anti-hCdc6 antibody (Santa CruzBiotechnology) (FIG. 4). UT: untreated cells; LP: treated with liposomewithout oligonucleotides; A: antisense oligonucleotides; M: mismatchedoligonucleotides. Each of the three active antisense oligonucleotideshC6-35a, hC6-39a, and hC6-60a reduced the hCdc6 protein level (Lanes 3,5 and 7).

Example 7

[0091] The Antisense Oligonucleotides hC45-30a with Anti-Cancer CellActivities Induced Activation of Caspase-3 and Cleavage of PARPIndicative of Apoptosis (Programmed Cell Death). The Chang's LiverCancer Cells were treated with the antisense oligonucleotides hC45-30a(A) (0.7 μM) or the corresponding mismatched (M) controloligonucleotides (see Table 1) in the same way as described in Example3. Proteins were extracted from the oligonucleotides-treated and controlcells and Western blotted with a polyclonal anti-caspase-3 antiserum ora monoclonal anti-PARP antibody (PharMingen). The antisenseoligonucleotides hC45-30a induced activation of caspase-3 (17 kD band;FIG. 5A, Lane 3) and cleavage of caspase-3 substrate PARP (86 kD band;FIG. 5B, Lane 3). Liposome (LP) without oligonucleotides (Lane 2) andthe mismatched oligonucleotides (Lane 4) also caused low levels ofactivation of caspase-3 and PARP cleavage, as they were slightlycytotoxic (see Table 1). UT: untreated cells; F.L. Casp-3: full-lengthcaspase-3.

Example 8

[0092] The Antisense Oligonucleotides hM2-7a Induced Cancer Cell Death.The Chang's Liver Cancer Cells were treated with the antisenseoligonucleotides hM2-47a (0.7 μM) or the corresponding mismatched(hM2-47m) or sense (hM2-47s) control oligonucleotides (see Table 1)conjugated with LipofectAMINE Plus (2%) in 70 l μof the Opti-MEM mediumwithout serum for 3 hrs. The cells were then grown in regular DMEMcontaining serum for 2 days before being photographed under an invertedlight microscope (FIG. 6). The antisense oligonucleotides hM2-47ainduced cell death of most of the cancer cells. UT: untreated cells; LP:liposome without oligonucleotides; A: antisense oligonucleotides; M:mismatched oligonucleotides; S: sense oligonucleotides.

Example 9

[0093] The Antisense Oligonucleotides Induced Apoptotic Cell Death. TheChang's Liver Cancer Cells were treated with an antisenseoligonucleotides (0.7 μM) or the corresponding sense controloligonucleotides conjugated with LipofectAMINE Plus (2%) in 70 μl of theOpti-MEM medium without serum for 3 hrs. The cells were then grown inregular DMEM containing serum for 4 hours before being harvested for DNAisolation and analysis on an agarose gel. The antisense oligonucleotidesinduced DNA fragmentation, which is an indication of apoptotic celldeath. M: DNA molecular weight markers; UT: untreated cells; LP:liposome without oligonucleotides; Sta: Staurasporine (as a positivecontrol for apoptosis); AS: antisense oligonucleotides; SS: sensecontrol oligonucleotides.

Example 10

[0094] The antisense oligonucleotides induced apoptosis in liver cancercells, but not in normal liver cells. The Chang's Liver Cancer Cells orthe L-02 normal liver cells were treated with an antisenseoligonucleotides (0.7 μM) conjugated with LipofectAMINE Plus (2%) in 70μl of the Opti-MEM medium without serum for 3 hrs. The cells were thengrown in regular DMEM containing serum for 4 hours before being analyzedby the TUNEL assay for DNA fragmentation, which is an indication ofapoptosis. The antisense oligonucleotides induced DNA fragmentation onlyin lever cancer cells, not in the normal liver cells.

Example 11

[0095] The antisense oligonucleotides reduced human cancer growth innude mice xenographs. Nude mice were inoculated with the HeLa tumorcells (by s.c. injection) and subsequently treated with the antisenseoligonucleotides (by i.v. injection; 2 μg/g body weight/day) for 13 days(indicated by the red arrow), three antisense oligonucleotides [hC3-35(#35a′), hM2-47 (m47a′) and hC45-18 (c18a′)] significantly reduced tumorgrowth compared to the control mice injected with the PBS buffer only.

[0096] Any reference to documents, acts, materials, devices, articles orthe like which has been included in the present specification is solelyfor the purpose of providing a context for the present claims and areincorporated by reference herein. It is not to be taken as an admissionthat any or all of these matters form part of the prior art and/ormodifications may be made to the invention as shown in the specificembodiments without departing from the spirit or scope of the inventionas broadly described. The present embodiments are, therefore, to beconsidered in all respects as illustrative and not restrictive.

1 27 1 16 DNA artificial sequence antisense oligonucleotide 1 aaggtgggaagttcaa 16 2 16 DNA artificial sequence antisense oligonucleotide 2aagatgggta ggtcaa 16 3 16 DNA human 3 ttgaacttcc cacctt 16 4 18 DNAartificial sequence antisense oligonucleotide 4 ctccctcttg gctcaagg 18 518 DNA artificial sequence antisense oligonucleotide 5 ctcccacctggttctagg 18 6 18 DNA human 6 ccttgagcca agagggag 18 7 19 DNA artificialsequence antisense oligonucleotide 7 agcctggcca acatggtaa 19 8 19 DNAartificial sequence antisense oligonucleotide 8 agccggacca gcattgtaa 199 19 DNA human 9 ttaccatgtt ggccaggct 19 10 16 DNA artificial sequenceantisense oligonucleotide 10 cttgaagacg ttgtgg 16 11 16 DNA artificialsequence antisense oligonucleotide 11 ctttaaggcg tagtgg 16 12 16 DNAhuman 12 ccacaacgtc ttcaag 16 13 16 DNA artificial sequence antisenseoligonucleotide 13 cagaaccagg gcccca 16 14 16 DNA artificial sequenceantisense oligonucleotide 14 cagcagcagg ccacca 16 15 16 DNA human 15tggggccctg gttctg 16 16 17 DNA artificial sequence antisenseoligonucleotide 16 tcccgcagat ggatgcg 17 17 17 DNA artificial sequenceantisense oligonucleotide 17 tccctcaggt ggaagcg 17 18 17 DNA human 18cgcatccatc tgcggga 17 19 20 DNA artificial sequence antisenseoligonucleotide 19 aggctgtcat ggagggacca 20 20 20 DNA artificialsequence antisense oligonucleotide 20 aggctctgag ggagtgacca 20 21 20 DNAhuman 21 tggtccctcc atgacagcct 20 22 19 DNA artificial sequenceantisense oligonucleotide 22 cgcgcatgtc cttcatccc 19 23 19 DNAartificial sequence antisense oligonucleotide 23 cgcgtatgcc catcttccc 1924 19 DNA human 24 gggatgaagg acatgcgcg 19 25 16 DNA artificial sequenceantisense oligonucleotide 25 gaagtgatct gtccct 16 26 16 DNA artificialsequence antisense oligonucleotide 26 gaggtgaact ttccct 16 27 16 DNAhuman 27 agggacagat cacttc 16

What is claimed is:
 1. An antisense oligonucleotides comprising asequence complementary to a target sequence encoding at least a portionof mRNA of a human replication-initiation gene, wherein theoligonucleotides inhibits the expression of replication-initiationprotein.
 2. The antisense oligonucleotides according to claim 1, whereinthe human replication-initiation gene is selected from the groupconsisting of hCdc6, hCdc45, hMcm2, hMcm3, hMcm4, hMcm5, hMcm6, hMcm7,hOrc1, hOrc2, hOrc3, hOrc4, hOrc5, hOrc6 and hCdt1.
 3. The antisenseoligonucleotides according to claim 2, wherein the gene is selected fromhCdc6, hCdc45, and hMcm2.
 4. The antisense oligonucleotides according toclaim 1, wherein the sequence is at least 90% complementary to thetarget sequence.
 5. The antisense oligonucleotides according to claim 4,wherein all or a portion of the sequence is selected from the groupconsisting of SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, and
 25. 6. Theantisense oligonucleotides according to claim 5, wherein the sequencecontains an 8 nucleotide base portion selected from the group consistingof SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, and
 25. 7. The antisenseoligonucleotides according to claim 6, wherein the sequence is selectedfrom the group consisting of SEQ ID NO. 1, 16 and
 25. 8. The antisenseoligonucleotides according to claim 1, wherein the oligonucleotidescontains a modified backbone or at least one modified linkage.
 9. Theantisense oligonucleotides according to claim 8, wherein theoligonucleotides is modified by phosphorothioate linkages.
 10. Theantisense oligonucleotides according to claim 9, wherein theoligonucleotides is modified by at least two phosphorothioate linkagessuch that each oligonucleotides contains a phosphorothioate linkagebetween the first two nucleotides and between the last two nucleotides.11. The antisense oligonucleotides according to claim 10, wherein theoligonucleotides contains at least one modified sugar moieties,nucleobases, or pharmacokinetic-enhancing moieties.
 12. The antisenseoligonucleotides according to claim 11, wherein the oligonucleotides isnuclease resistant.
 13. The antisense oligonucleotides according toclaim 1, wherein the target sequence is selected from the groupconsisting of SEQ ID NO. 3, 6, 9, 12, 15, 18, 21, 24, and
 27. 14. Acomposition comprising a chemotherapeutic agent and an antisenseoligonucleotides of claim 1 which inhibits expression of thereplication-initiation protein in cells.
 15. A pharmaceuticalcomposition comprising an effective amount of an antisenseoligonucleotides of claim 1, wherein expression of thereplication-initiation protein is inhibited.
 16. A method of treating orpreventing a hyperproliferative condition comprising administering to ahuman or cells thereof a therapeutically effective amount of antisenseoligonucleotides of claim 1, wherein expression of thereplication-initiation protein is inhibited.
 17. A method of diagnosingabnormal proliferative states comprising administering to a human orcells thereof an effective amount of antisense oligonucleotides of claim1 , wherein expression of the replication-initiation protein isinhibited.
 18. A method of determining human replication-initiationprotein expression comprising: (a) contacting human cells with aneffective amount of antisense oligonucleotides of claim 1 wherein theoligonucleotides inhibits the expression of human replication-initiationprotein; and (b) detecting the inhibition of the replication-initiationprotein.
 19. The method according to an of claims 16-18, wherein theantisense oligonucleotides is selected from the group consisting of SEQID NO. 1, 4, 7, 10, 13, 16, 19, 22, and
 25. 20. The method according toclaim 19 wherein the human replication-initiation gene is selected fromthe group consisting of hCdc6, hCdc45, hMcm2, hMcm3, hMcm4, hMcm5,hMcm6, hMcm7, hOrc1, hOrc2, hOrc3, hOrc4, hOrc5, hOrc6 and hCdt1. 21.The method according to claim 20, wherein the gene is selected from thegroup consisting of hCdc6, hCdc45, and hMcm2.
 22. The method accordingto claim 19, wherein the sequence contains an 8 nucleotide base portionselected from the group consisting of SEQ ID Nos. 1, 4, 7, 10, 13, 16,19, 22, and
 25. 23. The method according to claim 18, wherein theoligonucleotides contains a modified backbone or at least one modifiedlinkages.
 24. The method according to claim 23, wherein theoligonucleotides is modified by phosphorothioate linkages.
 25. Themethod according to claim 24, wherein the oligonucleotides is modifiedby at least two phosphorothioate linkages such that eacholigonucleotides contains a phosphorothioate linkage between the firsttwo nucleotides and between the last two nucleotides.
 26. The methodaccording to claim 25, wherein the oligonucleotides contains at leastone modified sugar moieties, nucleobases, or pharmacokinetic-enhancingmoieties.
 27. The method according to claim 26, wherein theoligonucleotides is nuclease resistant.
 28. The method according toclaim 16, wherein the target sequence is selected from the groupconsisting of SEQ ID NO. 3, 6, 12, 15, 18, 21, 24, and
 27. 29. A methodaccording to any of claims 16-18, further comprising: administering achemotherapeutic agent.
 30. A method of identifying antisenseoligonucleotides that inhibit expression of a gene: (a) screening anantisense oligonucleotides for inhibition of gene expression in vitro,wherein the oligonucleotides contains a phosphodiester DNA backbone; (b)selecting at least one antisense oligonucleotides from step (a) thatinhibit gene expression; (c) modifying at least two internucleosidiclinkages of the selected antisense oligonucleotides such that theoligonucleotides contains a phosphorothioate linkage between the firsttwo nucleotides and between the last two nucleotides of the sequence;(d) screening the modified oligonucleotides in step (c) for inhibitionof gene expression; (e) selecting at least one antisenseoligonucleotides from step (d) that inhibit gene expression; and (f)replacing one or more linkages of the oligonucleotides obtained fromstep (e) with phosphorothioate linkages.